connecting quorum sensing, c-di-gmp, pel …...quorum sensing signals, that it is in the periplasm,...

Connecting Quorum Sensing, c-di-GMP, PelPolysaccharide, and Biofilm Formation in Pseudomonasaeruginosa through Tyrosine Phosphatase TpbA(PA3885)Akihiro Ueda1, Thomas K. Wood1,2,3*

1 Artie McFerrin Department of Chemical Engineering, Texas A & M University, College Station, Texas, United States of America, 2 Department of Biology, Texas A & M

University, College Station, Texas, United States of America, 3 Zachry Department of Civil Engineering, Texas A & M University, College Station, Texas, United States of

America

Abstract

With the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing based on homoserine lactones was found toinfluence biofilm formation. Here we discern a mechanism by which quorum sensing controls biofilm formation byscreening 5850 transposon mutants of P. aeruginosa PA14 for altered biofilm formation. This screen identified the PA3885mutant, which had 147-fold more biofilm than the wild-type strain. Loss of PA3885 decreased swimming, abolishedswarming, and increased attachment, although this did not affect production of rhamnolipids. The PA3885 mutant also hada wrinkly colony phenotype, formed pronounced pellicles, had substantially more aggregation, and had 28-fold moreexopolysaccharide production. Expression of PA3885 in trans reduced biofilm formation and abolished aggregation. Wholetranscriptome analysis showed that loss of PA3885 activated expression of the pel locus, an operon that encodes for thesynthesis of extracellular matrix polysaccharide. Genetic screening identified that loss of PelABDEG and the PA1120 protein(which contains a GGDEF-motif) suppressed the phenotypes of the PA3885 mutant, suggesting that the function of thePA3885 protein is to regulate 3,5-cyclic diguanylic acid (c-di-GMP) concentrations as a phosphatase since c-di-GMPenhances biofilm formation by activating PelD, and c-di-GMP inhibits swarming. Loss of PA3885 protein increased cellular c-di-GMP concentrations; hence, PA3885 protein is a negative regulator of c-di-GMP production. Purified PA3885 protein hasphosphatase activity against phosphotyrosine peptides and is translocated to the periplasm. Las-mediated quorum sensingpositively regulates expression of the PA3885 gene. These results show that the PA3885 protein responds to AHL signalsand likely dephosphorylates PA1120, which leads to reduced c-di-GMP production. This inhibits matrix exopolysaccharideformation, which leads to reduced biofilm formation; hence, we provide a mechanism for quorum sensing control of biofilmformation through the pel locus and suggest PA3885 should be named TpbA for tyrosine phosphatase related to biofilmformation and PA1120 should be TpbB.

Citation: Ueda A, Wood TK (2009) Connecting Quorum Sensing, c-di-GMP, Pel Polysaccharide, and Biofilm Formation in Pseudomonas aeruginosa throughTyrosine Phosphatase TpbA (PA3885). PLoS Pathog 5(6): e1000483. doi:10.1371/journal.ppat.1000483

Editor: Frederick M. Ausubel, Massachusetts General Hospital, United States of America

Received December 12, 2008; Accepted May 22, 2009; Published June 19, 2009

Copyright: � 2009 Ueda, Wood. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from the National Institutes of Health (R01 EB003872) and the Army Research Office (W911NF-06-1-0408) to TKW,and by the Japan Society for the Promotion of Science as a postdoctoral fellowship to AU. The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Pseudomonas aeruginosa, an opportunistic pathogen, is often used

to elucidate how biofilms form because persistence of this

bacterium is linked to its ability to form biofilms [1]. Biofilms

are formed by the attachment of bacteria to submerged surfaces in

aquatic environments through their production of microbial

products including polysaccharides, proteins, and nucleic acids

[1]. In P. aeruginosa PA14, the glucose-rich extracellular polysac-

charide (EPS) of the biofilm matrix is formed by proteins encoded

by the pel operon; note the related strain P. aeruginosa PAO1 has

two EPS production loci, pel and psl [2,3]. Mutations in the pel

locus of P. aeruginosa PA14 dramatically decrease biofilm formation

as well as pellicle formation; pellicles are formed at the interface

between the air and liquid medium [3].

Regulation of Pel polysaccharide involves 3,5-cyclic diguanylic

acid (c-di-GMP) which is formed by diguanylate cyclases with

GGDEF motifs that synthesize this second messenger; phospho-

diesterases with EAL motifs catabolize c-di-GMP. Many proteins

with GGDEF motifs enhance biofilm formation [4]; for example,

c-di-GMP increases cellulose biosynthesis in Acetobacter xylinus [5],

and c-di-GMP enhances EPS production by binding the PelD

protein that is a c-di-GMP receptor in P. aeruginosa PA14 [6]. Thus,

biofilm formation is controlled by a signal cascade mediated by a

complex of c-di-GMP and PelD in P. aeruginosa PA14; however, the

upstream portions of this cascade have not been elucidated [7].

Quorum sensing (QS) is bacterial communication using

diffusible molecules known as autoinducers to regulate population

behavior and is related to both polysaccharide production and

biofilm formation. To date, three QS systems have been identified

PLoS Pathogens | www.plospathogens.org 1 June 2009 | Volume 5 | Issue 6 | e1000483

in P. aeruginosa. Las-based QS is regulated by N-(3-oxododecanoyl)-

L-homoserine lactone, produced by LasRI [8], and Rhl-based QS

is regulated by N-butyryl homoserine lactone, produced by RhlRI

[9]. The third QS molecule, 2-heptyl-3-hydroxy-4-quinolone

(PQS), was identified as a regulator for both Las- and Rhl-QS

[10]. These cell communication signals regulate several pheno-

types including virulence and antibiotic resistance [11]. Although

the relationship between QS and biofilm formation has not been

fully elucidated, some lines of evidence show the importance of QS

for biofilm formation. Cells lacking Las-QS in P. aeruginosa form

flat biofilms, and this structural abnormality makes bacteria in

biofilms more sensitive to antibiotic treatment [12]. Biofilm

architecture is regulated by rhamnolipids whose synthesis is

controlled by Rhl-QS [13]. Thus, P. aeruginosa QS seems to

participate in the development of biofilm architecture rather than

initiation of biofilm formation. In addition, LasR- and RhlR-QS

have been shown to influence the pel operons indirectly and

another transcriptional regulator that controls pel has been

predicted [7].

Protein phosphorylation and dephosphorylation are well-

conserved posttranslational modifications in both prokaryotes

and eukaryotes [14]. Protein kinases and phosphatases modulate

cellular activity by adding and removing phosphate groups at Ser,

Thr, or Tyr residues. Phosphorylation also occurs at His and Asp

residues by histidine kinases and response regulators in two-

component regulatory systems. Although the discovery of protein

phosphorylation was delayed in prokaryotes compared to

eukaryotes [14], many genome sequences predict the existence

of phosphorylation/dephosphorylation systems in prokaryotes.

The P. aeruginosa genome encodes an extraordinary number of the

genes for two-component regulatory systems [15], and diverse

cellular functions are regulated by His-Asp phosphorylation

including chemotaxis, iron acquisition, alginate production, and

virulence factors [16]. In contrast, phosphorylation at Ser, Thr,

and Tyr residues has not been studied well in P. aeruginosa;

although, Fha1 of the Type VI secretion system is posttransla-

tionally regulated through Thr phosphorylation by the protein

kinase PpkA and dephosphorylated by the phosphatase PppA [17].

In Bacillus subtilis, mutations in prkC, a Ser/Thr kinase, and prpC, a

phosphatase, decrease sporulation and biofilm formation [18].

Mutations in stk1, a Ser/Thr kinase, and stp1, a phosphatase of

Stk1, decrease virulence in Streptococcus agalactiae [19]. These

findings show that posttranslational modification via protein

phosphorylation at Ser, Thr, and Tyr residues regulates various

cellular functions.

In this study, our goal was to explore the complex regulatory

cascade that includes detection of QS signals, Pel polysaccharide

production, and biofilm formation. By screening 5850 transposon

mutants for altered biofilm formation, we identified and

characterized a novel protein tyrosine phosphatase, TpbA

(tyrosine phosphatase related to biofilm formation), that represses

biofilm formation through the pel locus. The tpbA mutant displays

pleiotropic phenotypes such as hyperbiofilm formation, enhanced

EPS production, altered colony morphology, increased aggrega-

tion, elevated c-di-GMP, and abolished swarming. Loss of an

uncharacterized GGDEF protein, PA1120 (TpbB), suppressed

these phenotypes, indicating that TpbA controls c-di-GMP

production through TpbB. Therefore, the mechanism for QS-

control of biofilm formation has been extended to include a novel

phosphatase (TpbA), a diguanylate cyclase (TpbB), and c-di-GMP;

hence, the predicted additional level of control of the pel

polysaccharide locus has been identified and involves c-di-GMP

as controlled by a tyrosine phosphatase.

Results

TpbA negatively regulates biofilm formation andpositively regulates swimming and swarming

Previously, by screening 5850 transposon mutants for altered

biofilm formation, we identified 137 transposon mutants of P.

aeruginosa PA14 with over 3-fold enhanced biofilm formation [20].

Among these mutants, the tpbA (PA3885) mutant increased biofilm

formation by 147-fold after 8 h in LB medium at 37uC (Fig. 1A).

This significant increase in biofilm formation upon inactivating tpbA

is partially due to enhanced attachment to the polystyrene surface

because biofilm formation at the bottom of the plates (solid/liquid

interface) increased gradually with the tpbA mutant while PA14 did

not form biofilm on the bottom of the plate (Fig. 1B).

Motility often influences biofilm formation in P. aeruginosa; biofilm

formation is inversely influenced by swarming motility [21], and

swimming motility increases initial attachment to surfaces during

biofilm development [22]. To examine the relationship between

enhanced biofilm formation and motility in the tpbA mutant, we

examined swimming and swarming motility for this mutant; rhlR

[23] and flgK [22] mutants were used as negative controls for

swarming and swimming motility, respectively. Although PA14

swarmed on the surface of plates at 24 h, the tpbA mutations

abolished swarming like the rhlR mutation (Fig. 2A). The tpbA

mutation also decreased swimming motility by 40% (Fig. 2B).

Swarming is positively influenced by production of the biosurfactant

putisolvins in P. putida [24] and rhamnolipids in P. aeruginosa [23].

However, no significant difference was found in the production of

rhamnolipids between PA14 and the tpbA mutant (Fig. 2C). This

shows the tpbA mutation abolishes swarming in a manner distinct

from the production of rhamnolipids.

TpbA affects colony morphology, decreases EPS, anddecreases pellicle production

Congo-red is often used to observe colony morphology because

it detects EPS production and this impacts biofilm formation; for

example, the wspF mutant shows wrinkly colony morphology on

Author Summary

Most bacteria live in biofilms, which are complexcommunities of microorganisms attached to a surface viapolysaccharides; these biofilms are responsible for mosthuman bacterial diseases. The pathogen Pseudomonasaeruginosa is best-studied for biofilm formation. Currently,it is recognized that cell communication or quorumsensing is important for biofilm formation, but how theseexternal signals are converted into internal signals toregulate the networks of genes that result in biofilmformation is not well understood. Here, by studying 5850bacterial strains, each of which lacks a single protein, weidentify a new enzyme of P. aeruginosa, a tyrosinephosphatase (TpbA), that links extracellular quorumsensing signals to polysaccharide production and biofilmformation. We find that TpbA is subject to control byquorum sensing signals, that it is in the periplasm, and thatit controls the level of the intracellular secondarymessenger 3,5-cyclic diguanylic acid (c-di-GMP). By con-trolling c-di-GMP concentrations, TpbA serves to regulatebiofilm formation, rapid cell movement on the surface,colony morphology, cell aggregation, and polysaccharideproduction. The importance of our study is that it showsthe secondary messenger c-di-GMP may be regulated bytyrosine phosphorylation; hence, it provides a new targetfor controlling bacterial social behavior.

TpbA Reduces Biofilm via c-di-GMP


Congo-red plates and has increased biofilm formation [25], while

smooth colonies like the pelA mutant [3] form less biofilm. We

found that the tpbA mutant formed a red, wrinkly colony when it

was grown on Congo-red plates at 37uC, although PA14 and the

pelA mutant formed white smooth colonies (Fig. 3A). When the

bacteria were grown at 25uC, both PA14 and the tpbA mutant

formed red wrinkly colonies, but the pelA mutant still formed a

white smooth colony (Fig. 3A). These observations with the pelA

mutant and wild-type PA14 are identical to the previous report

that expression of the pel genes is induced at room temperature

and repressed at 37uC [7]. Therefore, the red wrinkly colony

formed by the tpbA mutant at 37uC implies increased production

of EPS via Pel.

We also quantified the amount of EPS bound to cells of PA14

and the tpbA mutant at both 37uC and 25uC. As shown in Fig. 3B,

the tpbA mutant produced 28-fold more EPS than PA14 at 37uC.

The tpbA mutant also produced 4.3-fold more EPS than PA14 at

room temperature. The pelA mutant (negative control) did not

form EPS at both temperatures tested. We also found that the tpbA

mutant formed a pronounced pellicle at 37uC after 1 day, but

PA14 and the pelA mutant did not form a pellicle (data not shown).

At 25uC, both the tpbA mutant and PA14 formed pellicles after 5

days. Taken together with the EPS production data, TpbA

reduces pellicle formation by decreasing Pel activity.

Differentially regulated genes in biofilm cells of the tpbAmutant

To confirm the impact of the tpbA mutation on pel expression

and to investigate its impact on the whole genome, a whole-

transcriptome analysis was performed with biofilm cells of the tpbA

mutant at 37uC at 7 h; planktonic cells were not assayed since we

were primarily interested in how TpbA controls biofilm formation.

Inactivation of tpbA altered diverse loci including genes related to

EPS production (pelACDF induced approximately 4-fold), transport

(PA2204 repressed approximately 5-fold, PA4142–PA4143 in-

duced approximately 3-fold), type IV pili (PA4302 to PA4306

repressed approximately 4-fold), and a putative adhesin and its

regulator (PA4624–PA4625 induced approximately 4-fold) (Tables

S1 and S2). Expression of tpbA was induced as much as 120-fold in

the tpbA mutant, suggesting that TpbA negatively regulates its

transcription. The whole-transcriptome experiments were per-

formed twice using independent cultures of PA14 and the tpbA

mutant at 7 h, and most of the differentially regulated genes were

consistently altered except pel genes which were induced the most

in the samples containing an RNase inhibitor. A whole-

transcriptome analysis was also conducted using biofilm cells at

4 h since the mode of growth switched from planktonic to biofilm

for the tpbA mutant at this time (Fig. 1A). Similar to the 7 h results,

several loci were induced including pelAEF (1.5- to 1.7-fold), tpbA

(42-fold), PA1168–PA1169 (1.4- to 2.1-fold), PA3886 (3.5-fold),

and PA4624–PA4625 (2- to 3.7-fold) (Table S1).

To verify induction of the pel locus, expression of pelA was

determined by quantitative real time-PCR (qRT-PCR). Using two

independent RNA samples extracted from biofilm cells at 7 h, pelA

was induced 1126100-fold in the tpbA mutant vs. PA14. These

results showed EPS production is induced significantly in the tpbA

mutant due to overexpression of pel genes. qRT-PCR also

confirmed induction of PA4625 (767-fold) as well as PA4139

(38630-fold) that encodes a hypothetical protein.

TpbA represses adhesin expression and reducesaggregation

Cell aggregative behavior is also related to biofilm formation so

we investigated the role of TpbA on cell aggregation and found the

tpbA mutant causes cell aggregation (Fig. 4A). Autoaggregation of

the tpbA mutant was also observed in 96-well polystyrene plates

during biofilm formation (data not shown). Our whole-transcrip-

tome analysis showed that inactivating tpbA induced both PA4624

(encodes for a putative hemolysin activator) and PA4625 (encodes

for an adhesin/hemagglutinin) by 2.1- to 4.9-fold. In E. coli,

adhesin regulates cell aggregation as well as attachment [26]. To

examine whether PA4624–PA4625 control adhesive activity in P.

aeruginosa, we investigated biofilm formation with these mutants.

Both mutants showed decreased initial biofilm formation; i.e.,

initial attachment, to polystyrene plates at 1 h and 2 h (Fig. 4B),

and final biofilm formation at 24 h was also decreased for both the

PA4624 and PA4625 mutants, which suggests that both gene

products control attachment to the surface. Therefore, TpbA

decreases cell aggregation probably by repressing the PA4624 and

PA4625 genes.

Figure 1. Inactivation of tpbA increases biofilm formation. Total biofilm formation (at the liquid/solid and air/liquid interfaces) (A), and biofilmformation on the bottom of polystyrene plates (B) by P. aeruginosa PA14 and the tpbA mutant at 37uC in LB after 50 h. Six to ten wells were used foreach culture. Data show the average of the two independent experiments6s.d.doi:10.1371/journal.ppat.1000483.g001



Complementation of the tpbA mutationTo verify whether the phenotypes observed in the tpbA mutant

were caused by loss of function of TpbA, we confirmed transposon

insertion in tpbA by PCR at residue 25. Furthermore, biofilm

formation for both PA14 and the tpbA mutant were examined with

tpbA expressed in trans under the control of an arabinose-inducible

promoter. tpbA expression reduced biofilm formation of the tpbA

mutant by 33% (Fig. S1A) and abolished biofilm formation on the

bottom of the plates (Fig. S1B). Similar results were found upon

expressing tpbA in wild-type PA14 (OD540 value was 0.2260.02 for

PA14/pMQ70 and 0.0260.01 for PA14/pMQ70-tpbA, Fig. S1A).

In addition, the aggregative phenotype of the tpbA mutant was also

complemented by expression of tpbA in trans (Fig. S1C). Taken

together, TpbA functions as a negative regulator of biofilm

formation and aggregation in PA14.

Genetic screening identified Pel and GGDEF-proteinsdownstream of TpbA

To investigate how TpbA regulates biofilm formation, EPS

production, wrinkly colony morphology, and cell aggregation, genetic

screening was conducted using Tn5-luxAB transposon mutagenesis to

find suppressive loci for the phenotypes of the tpbA mutation. The

double mutant library (tpbA plus random gene inactivations) was

screened first for a reduction in aggregation; this step eliminated most

cells with unaltered phenotypes by allowing them to aggregate and

precipitate at the bottom of the tube. The cells remaining in the

supernatant that failed to aggregate like the tpbA mutant were grown

on Congo-red plates, incubated at 37uC for 3–4 days, and colonies

displaying a white and smooth shape like the wild-type strain were

chosen. After that, a third screen was performed by assaying biofilm

Figure 2. TpbA regulates swarming, swimming motility, andproduction of rhamnolipids. Swarming motility (A), swimmingmotility (B), and production of rhamnolipids (C) of P. aeruginosa PA14and the tpbA mutant at 37uC after 24 h. Five plates were used for eachswarming and swimming culture, and data show the average of twoindependent experiments. For the production of rhamnolipids, datashow the average of the two independent experiments6s.d.doi:10.1371/journal.ppat.1000483.g002

Figure 3. Inactivation of tpbA increases colony roughness andenhances EPS production. Colony morphology of P. aeruginosaPA14, the tpbA mutant, and the pelA mutant on Congo-red plates after6 days at 25uC or 37uC (A). EPS production of each strain after 24 h at37uC or after 48 h at 25uC (B). Data show the average of the twoindependent experiments6s.d.doi:10.1371/journal.ppat.1000483.g003



formation using 96-well polystyrene plates to identify double mutants

that had biofilm formation like the wild-type strain. Twenty-six

mutants were identified that showed reduced aggregation, a white

smooth colony, and reduced biofilm formation like the wild-type

strain, and 19 of these mutations were in the pel locus (Fig. 5A,

Table 1). Four of the other mutants have the Tn5-luxAB insertion in

the TpbB gene (encodes a GGDEF-motif protein) and in the PA1121

gene (encodes a hypothetical protein). In addition, insertions were

found in the PA1678 gene (encodes a putative DNA methylase) and

in the promoter of the PA5132 gene (encodes a putative protease)

(Table 1). Like the double mutants, all of the single mutants lacking

the gene identified by genetic screening were tested for biofilm

formation, and all of these mutants formed less biofilm as reported

previously (Fig. 5) [3,4].

TpbA negatively controls cellular c-di-GMPconcentrations

Results of genetic screening and the whole-transcriptome analysis

implied TpbA regulates c-di-GMP concentrations since loss of one of

the GGDEF proteins (TpbB) masked the phenotypes of the tpbA

mutant. tpbB encodes a functional GGDEF protein whose activity

was confirmed by overexpressing this gene in P. aeruginosa [4]. We also

confirmed that expression of tpbB increases cell aggregation and

attachment to tubes so the tpbB mutation may be complemented (Fig.

S2). In addition, we measured the cellular c-di-GMP concentrations

of PA14 and the tpbA mutant using high performance liquid

chromatography (HPLC) as reported previously [4]. The peaks

corresponding to c-di-GMP were observed with the extracts of the

tpbA mutant, but not with those of PA14, and the peak was confirmed

by comparing the spectrum to purified c-di-GMP as well as by spiking

the samples with purified c-di-GMP (Fig. S3). We estimated the

cellular c-di-GMP concentration was 1062 pmol/mg cells in the

tpbA mutant. This is comparable to the c-di-GMP concentration

found for a small colony variant that showed aggregation (around

2.0 pmol/mg cells) and a mutant with wrinkly colony morphology

[27]. Overexpression of tpbB results in c-di-GMP concentrations of

134 pmol/mg cells in PA14 [4]. Therefore, TpbA reduces c-di-GMP

concentrations in the cell and probably does so via TpbB.

Figure 4. Inactivation of tpbA increases cell aggregation. Aggregation of PA14 and the tpbA mutant after diluting with fresh LB medium(percentages indicate volume % of the starting overnight culture and fresh medium) (A). Biofilm formation of mutants lacking adhesin (PA4625) and itsregulator (PA4624) at 37uC after 1, 2, and 24 h (B). Ten wells were used for each culture. Data show the average of the two independent experiments6s.d.doi:10.1371/journal.ppat.1000483.g004



TpbA is a tyrosine phosphatasetpbA encodes a 218 aa protein that has the conserved domain for

a protein tyrosine phosphatase [28,29] since it has the C(X)5R(S/

T) motif beginning at aa 132 (CKHGNNRT). To confirm it is a

tyrosine phosphatase, we purified TpbA by adding a polyhistidine

tag at either the N-terminus (TpbA-nHis) or the C-terminus

(TpbA-cHis) (note only the C-terminus fusion protein was active).

Expression of recombinant TpbA was confirmed in E. coli by clear

expression of a band at 24 kD (Fig. 6A). The purified TpbA

protein had phosphatase activity with p-nitrophenyl phosphate

(pNPP) that is often used as a general phosphatase substrate [29]

(Fig. 6B). Further proof that TpbA is a tyrosine phosphatase was

found using a tyrosine phosphatase specific inhibitor, trisodium

orthovanadate [30], that completely inhibited the phosphatase

activity of TpbA-cHis (Fig. 6B). The third and fourth lines of

evidence that TpbA is a tyrosine phosphatase were found using

tyrosine specific substrates; TpbA-cHis dephosphorylated both

phosphotyrosine peptides, END(pY)INASL (peptide type I) and

DADE(pY)LIPQQG (peptide type II) (Fig. 6C), and this activity

was inhibited by trisodium orthovanadate. These results show

conclusively that TpbA encodes a tyrosine phosphatase.

Tyrosine phosphorylation enhances biofilm formation inPA14

To see the effect of tyrosine phosphorylation on biofilm

formation, biofilm formation was examined in PA14 with

trisodium orthovanadate at 37uC for 4 h which should reduce

dephosphorylation by TpbA. Trisodium orthovanadate increased

PA14 biofilm formation 3.6-fold (Fig. S4), showing that cellular

tyrosine phosphorylation increases biofilm formation.

TpbA is found in the periplasmThe N-terminal region of TpbA protein has a putative signal

peptide, predicted by pSORT [31], that appears necessary for

secretion of this protein (28 aa, MHRSPLAWLRLLLAAVL-

GAFLLGGPLHA). This implied that processing of N-terminal

region of TpbA protein may be essential for full phosphatase

activity. To prove that TpbA has an active signal sequence, we

expressed TpbA in E. coli and collected the proteins from cytosolic,

periplasmic, and membrane fractions. All fractioned proteins were

analyzed by SDS-PAGE, and we found that TpbA exclusively

localized in the periplasm (data not shown). Hence, TpbA

probably dephosphorylates its substrate in the periplasm which

explains why phosphatase activity was seen only with the fusion

protein with the His tag at the C-terminus.

Y48 and Y62 are responsible for TpbB activitySince TpbA is a tyrosine phosphatase that is found in the

periplasm and since TpbB has three likely periplasmic tyrosines

(Y48, Y62, and Y95) [32], we mutated the periplasmic tyrosine

residues by converting them to phenylalanine and checked for

TpbB activity in the tpbB mutant. Active TpbB, from overexpres-

sion of tpbB using the tpbB mutant and pMQ70-tpbB, always leads

to aggregation whereas the empty plasmid does not cause

aggregation (Fig. S5); hence, if a necessary tyrosine is mutated,

there should be a reduction in aggregation. Aggregation was

always observed with TpbB-Y95F in nine cultures; hence TpbB-

Y95F remains active even though it lacks tyrosine 95 so this

tyrosine is not phosphorylated/dephosphorylated. In contrast, the

Y48F mutation of TpbB decreased aggregation for 43% of the

cultures (20 of 46 cultures did not aggregate), and the Y62F

mutation decreased aggregation for 24% of the cultures (9 of 37

cultures did not aggregation). Hence, both Y48 and Y62 are likely

targets for tyrosine phosphorylation/dephosphorylation of TpbB

with Y48 preferred. We confirmed that these mutations did not

affect expression level of TpbB protein (data not shown).

TpbA tyrosine phosphatase is unique among bacteriaand eukaryotes

Tyrosine phosphorylation and dephosphorylation have crucial

roles in cellular signaling and are well-conserved among many

organisms [33]. Some bacterial tyrosine phosphorylations have

been identified and these regulatory systems control divergent

cellular functions [34]. In order to predict whether TpbA function

is conserved among other species, we conducted a BLASTP search

and found the TpbA protein is highly conserved among P.

aeruginosa (PAO1, PA14, C3719, and PA7 with an E value less than

Figure 5. Reduction in biofilm formation by tpbA phenotypereversal mutations. Biofilm formation of double mutants (A) andsingle mutants (B) identified by genetic screening for the tpbA mutationat 37uC in LB after 24 h. Six to ten wells were used for each culture.Representative data are shown in (A). Biofilm formation of each mutantwas calculated relative to that of PA14 (OD540 mutant/OD540 wild-type).Data show the average of the two independent experiments6s.d.doi:10.1371/journal.ppat.1000483.g005



3e-98) and is well-conserved among P. fluorescens Pf-5, P. fluorescens

Pf0-1, P. mendocina, Burkholderia cepacia, Pelobacter carbinolicus,

Desulfatibacillum alkenivorans, Bacteroides thetaiotaomicron, B. ovatus, B.

caccae, Acinetobacter baumannii, Desulfococcus oleovorans, and Geobacter

metallireducens (E values less than 3e-11). All of these conserved

tyrosine phosphatases have the C(X)5R(S/T) signature and most

are uncharacterized. Even though protein similarity is not very

high, some eukaryotes, such as Homo sapiens and Arabidopsis thaliana,

Table 1. Phenotype reversal loci for the tpbA mutation.

Strain PAO1 ID PA14 IDGeneName Gene function

Relativebiofilmformation

Colonymorphology Aggregation

PA14 - - - - 0.14 white smooth 2

tpbA PA3885 PA14_13660 tpbA Tyrosine phosphatase (this study) 1.00 red wrinkly +

Mutant 1 PA1120 PA14_49890 tpbB c-di-GMP cyclase, GGDEF motif 0.13 white smooth 2

Mutant 19 PA1120 PA14_49890 tpbB c-di-GMP cyclase, GGDEF motif 0.10 white smooth 2

Mutant 4 PA1121 PA14_49880 Hypothetical protein 0.22 white smooth 2

Mutant 11 PA1121 PA14_49880 Hypothetical protein 0.07 white smooth 2

Mutant 2 PA1678 PA14_42790 Putative DNA methylase 0.30 white smooth 2

Mutant 6 PA1678 PA14_42790 Putative DNA methylase 0.28 white smooth 2

Mutant 5 PA3058 PA14_24560 pelG Predicted membrane protein relatedto EPS production, PelG

0.24 white smooth 2


0.19 white smooth 2


0.27 white smooth 2


0.25 white smooth 2


0.42 white smooth 2

Mutant 10 PA3060 PA14_24530 pelE Sucrose synthase related to EPSproduction, PelE

0.41 white smooth 2

Mutant 15 PA3060 PA14_24530 pelE Sucrose synthase related to EPSproduction, PelE

0.45 white smooth 2

Mutant 3 PA3061 PA14_24510 pelD L-lactate permease related to EPSproduction, PelD

0.20 white smooth 2

Mutant 16 PA3063 PA14_24490 pelB Conserved hypothetical proteinrelated to EPS production, PelB

0.31 white smooth 2


0.21 white smooth 2


0.17 white smooth 2


0.38 white smooth 2


0.28 white smooth 2


0.23 white smooth 2

Mutant 8 PA3064 PA14_24480 pelA Oligogalacturonide lyase relatedto EPS production, PelA

0.18 white smooth 2


0.45 white smooth 2


0.29 white smooth 2

Mutant 20 PA3064 Pro-PA14_24480 pelA Oligogalacturonide lyase relatedto EPS production, PelA

0.25 white smooth 2


0.24 white smooth 2

Mutant 13 PA5132 Pro-PA14_67780 Putative protease 0.15 white smooth 2

Genetic screening identified additional mutations that mask the phenotypes of the tpbA mutant (enhanced biofilm formation, EPS production, wrinkly colonymorphology, and aggregation). Relative biofilm formation is shown as a ratio of that of the tpbA mutant.doi:10.1371/journal.ppat.1000483.t001



have TpbA homologs with a C(X)5R(S/T) signature. Therefore,

TpbA and TpbA homologs may share important functions in

procaryotes and eucaryotes.

Las-QS regulates expression of tpbAQS regulates many genes in P. aeruginosa via a conserved cis-

element in the promoter of each gene. N-(3-oxododecanoyl)-L-

homoserine lactone binds to the LasR transcriptional regulator

[35], and this complex interacts with the las-box, defined as CT-

(N)12-AG sequence [36]. The Las-box is conserved among the

promoters of the Las-QS regulated genes including lasB, rhlAB,

and rhlI [36]. Another class of transcriptional regulation is

governed by the lys-box, that is defined as a palindromic sequence,

T-(N)11-A [37], and MvfR is a LysR-type transcription factor that

binds to the lys-box [38]. We found that the promoter of tpbA

(ptpbA) has a putative las-box 220 bp upstream of the start codon

(CTCGCCTCGCTGAAAG) and a putative lys-box 90 bp

upstream of the start codon (TGAAGCTGCCTCA). In order to

examine if expression of tpbA is regulated by QS, we constructed a

ptpbA::lacZ fusion plasmid (pLP-ptpbA) and transformed this into

QS-related PA14 mutants (lasI, rhlI, and lasR rhlR). Expression of

tpbA gene in biofilm cells was reduced by 42% in the lasI mutant,

but not in the rhlI mutant (Fig. 7). Corroborating these results,

inactivation of both lasR and rhlR also decreased expression of tpbA

gene by 39% (Fig. 7). Similar results were obtained when the

activity of ptpbA::lacZ was examined in planktonic cells (50%

reduction in transcription for the lasI mutant and 37% reduction

for the lasR rhlR mutant). Since loss of QS only affected expression

of tpbA by 50%, other factors may also participate in the regulation

of tpbA. These results suggest that Las-QS, rather than Rhl-QS, is

an activator of tpbA expression with other unknown regulators.

We also investigated whether the tpbA mutation influences the

regulation of Las- and Rhl-QS using plasR::lacZ and prhlR::lacZ

plasmids. Expression of lasR was slightly increased (1.3-fold) with

the tpbA mutation. This indicated that Las-QS has more impact on

expression of tpbA than tpbA does on that of Las-QS. In addition,

expression of rhlR was decreased by 2-fold in the tpbA mutant.

Hence, LasR appears to enhance tpbA transcription and TpbA

leads to increased rhl transcription.

Discussion

In this study, we demonstrate that TpbA is a tyrosine

phosphatase that regulates diverse phenotypes in P. aeruginosa

including the concentration of cellular c-di-GMP. As a second

messenger, c-di-GMP is a positive regulator of biofilm formation

[4], EPS production [6], and pellicle formation [4], and a negative

regulator of swarming motility [39]. The lines of evidence that

show TpbA represses c-di-GMP production in P. aeruginosa that we

found are (i) inactivating tpbA increases c-di-GMP (Fig. S3); (ii)

inactivating tpbA increases biofilm formation (Fig. 1), EPS

production (Fig. 3B), and pellicle formation, and c-di-GMP

stimulates biofilm formation [4], EPS production [6], and pellicle

Figure 6. TpbA has phosphatase activity against tyrosine residues. Purification of TpbA-cHis (lane 1: protein marker, lane 2: whole cell lysatefrom E. coli BL21(DE3)/pET28b-13660c after 3 h of IPTG induction, lane 3: purified TpbA-cHis) (A). p-Nitrophenyl phosphate phosphatase assay withTpbA-cHis protein (B). Phosphatase reaction was performed at 37uC for 1 h with the indicated amount of protein. Na3VO4 (10 mM) was used as aninhibitor specific for tyrosine phosphatases. Protein tyrosine phosphatase assay with TpbA-cHis (C). Phosphatase reaction was performed withsynthetic phosphotyrosine peptides (type I: END(pY)INASL and type II: DADE(pY)LIPQQG) at 37uC for 3 h. Na3VO4 (50 mM) was used as an inhibitor.doi:10.1371/journal.ppat.1000483.g006

Figure 7. Las QS activates transcription of tpbA. b-galactosidaseactivity of ptpbA was measured with biofilm cells of PA14 and themutants lasI, rhlI, and lasR rhlR using pLP-ptpbA. Data show the averageof the two independent experiments6s.d.doi:10.1371/journal.ppat.1000483.g007



formation [4]; (iii) inactivating tpbA increases expression of the pel

locus (seen via the whole-transcriptome analysis and RT-PCR),

and c-di-GMP activates expression of pelA [6]; (iv) inactivating tpbA

increases aggregation (Fig. 4) and expression of adhesins (PA4625),

and c-di-GMP stimulates adhesion [40]; (v) inactivating tpbA

decreases motility (abolishing swarming and decreasing swimming

in the tpbA mutant, Fig. 2AB), and c-di-GMP decreases swarming

[40]; (vi) inactivating tpbB (encodes a GGDEF-motif protein that

produces c-di-GMP [4]) suppresses the phenotypes observed in the

tpbA mutant, and (vii) expression of tpbA and tpbB in trans

complements aggregation/biofilm formation and aggregation,

respectively. Thus, TpbA represses these phenotypes by decreasing

c-di-GMP. A proposed regulatory mechanism for biofilm

formation by TpbA is shown in Fig. 8.

EPS production in P. aeruginosa PA14 is regulated by PelA [3].

Transcription of pelA is higher at temperatures lower than 37uC,

and PA14 forms more biofilm at lower temperatures [7].

However, the tpbA mutation seems to constitutively enhance pel

expression independently from this temperature regulation as seen

in the enhanced EPS production at 37uC (Fig. 3) and the whole-

transcriptome analysis that was conducted at 37uC (Table S2). In

addition to increased expression of pelA, additional activation of

Pel proteins might be caused by the increased c-di-GMP

concentration by the tpbA mutation since c-di-GMP binds PelD

and increases EPS production [6]. In addition to the enhanced

EPS production (Fig. 3B) and increased pel expression (Fig. 3A,

Table S1, and qRT-PCR) seen in the tpbA mutant, another reason

why inactivating tpbA increased biofilm formation is the elevated

adhesin activity as seen via enhanced biofilm formation on the

bottom of polystyrene plates (Fig. 1B). Cell surface adhesins affect

bacterial adhesive activity [41], and we have discovered a novel

adhesin (PA4625) that is related to TpbA (Table S1) and to initial

biofilm formation (Fig. 4B). Since expression of adhesion factors is

also positively regulated by c-di-GMP [40], elevated c-di-GMP

level enhances adhesion of the tpbA mutant.

c-di-GMP seems to control the switch of motility-sessility of the

tpbA mutant since inactivation of TpbA abolished swarming

motility (Fig. 2A) and decreased swimming motility by 40%

(Fig. 2B), although regulation of swarming motility is very complex

as its activity is controlled by QS, flagellar synthesis, and

production of rhamnolipids [42]. In addition, our whole-

transcriptome results showed weak repression of some of flagellar

biosynthesis genes (flg, fle, and fli loci) due to the elevated c-di-

GMP, and activity of FleQ, a transcriptional activator of flagellar

biosynthesis, is repressed upon binding c-di-GMP [43]. Hence, the

increased c-di-GMP concentrations may repress motility of the

tpbA mutant via the FleQ pathway that affects expression of

flagellar synthesis genes.

Many genes are expected to be differentially regulated by

changing c-di-GMP concentrations since it plays a role as a second

Figure 8. Schematic of TpbA regulation of biofilm formation in P. aeruginosa PA14. The QS molecule, N-(3-oxododecanoyl)-L-homoserinelactone (3-oxoC12-HSL), binds to the LasR transcription factor, and this complex activates expression of tpbA. TpbA has a N-terminal signal sequenceand is translocated into the periplasm. Periplasmic TpbA dephosphorylates the membrane-anchored GGDEF protein TpbB at a tyrosine reside whichdeactivates GGDEF protein activity. The reduced cellular c-di-GMP concentration decreases expression of the pel operon as well as adhesin genes.This leads to reduced EPS production, biofilm formation, and pellicle formation, as well as enhanced swarming motility. Production of rhamnolipids isnot regulated by TpbA.doi:10.1371/journal.ppat.1000483.g008



messenger in P. aeruginosa. Similar regulation of gene expression was

observed between the tpbA mutant and the other strains related to c-

di-GMP production. For example, production of PA1107, TpbB,

and PA3702 proteins that have a GGDEF-domain leads to

activation of pelA expression [6]. Mutation in wspF, encoding a

CheB-like methylesterase, increases both biofilm formation and c-

di-GMP production [25]. wspF mutation altered expression of genes

such as pelABCDEFG, PA4624, PA4625, PA2440, and PA2441

whose expression are induced in the tpbA mutant (Table S2).

Common regulation of these genes may be partially controlled by

the elevated cellular c-di-GMP concentrations. In contrast,

expression of pelA was not induced in the bifA mutant that produces

more c-di-GMP [44]. This may be because regulation of c-di-GMP

signaling is complex in that the P. aeruginosa genome encodes 17

diguanylate cyclases, 5 phosphodiesterases, and 16 diguanylate

cyclase-phosphodiesterase proteins [4].

Relevance of c-di-GMP to regulation of diverse cellular functions

is now an emerging topic in bacteriology. This second messenger is

an activator of cellulose synthase in Acetobactor xylinum [5] and

controls many phenotypes in P. aeruginosa [4]. Several GGDEF

proteins for synthesis and EAL proteins for degradation of c-di-

GMP have been identified in P. aeruginosa [6,39,44], and increased

production of c-di-GMP enhances biofilm formation and decreases

swarming motility [4,6,39,44]. Similarly, enhanced biofilm forma-

tion and/or abolished swarming motility were observed in the tpbA

mutant via increased production of cellular c-di-GMP. Since TpbA

does not possess GGDEF and EAL domains, this protein indirectly

influences cellular c-di-GMP concentrations via its phosphatase

activity as shown by activity with both pNPP, a broad substrate for

phosphatases, and two phosphotyrosine-specific peptides (Fig. 6).

We also found that processing the N-terminal signal sequence may

be necessary for TpbA activity in the periplasm. Hence, our results

reveal a novel regulatory mechanism for cellular c-di-GMP

concentration by tyrosine phosphorylation in the periplasm of P.

aeruginosa; control of c-di-GMP by tyrosine phosphorylation has not

been shown previously.

There is little known about the regulation of GGDEF and EAL

proteins in regard to regulation of c-di-GMP level. A chemosen-

sory system, encoded by wspABCDEFR in PAO1, regulates c-di-

GMP production via a His-Asp phosphorylation relay [25]. For

the tpbA mutant, tpbB was found to reverse the phenotype of tpbA,

suggesting that overproduction of c-di-GMP is clearly related to

the phenotypes of the tpbA mutant as overexpression of this gene

caused pronounced aggregation (Fig. S2). Probably, TpbB, or

another GGDEF protein, might participate in c-di-GMP synthesis

in the tpbA mutant. A comprehensive analysis of all of the P.

aeruginosa GGDEF proteins has been completed, and those

GGDEF proteins that abolished or decreased biofilm formation

are PA0169, PA1107, TpbB, PA1181, PA1433, PA1727, PA3702,

PA4959, and PA5487 [4]. Among these GGDEF proteins, only

PA1107, TpbB, PA3702, and PA5487 increased biofilm formation

when their genes were overexpressed [4]. Because TpbA is a

periplasmic protein, its target GGDEF protein should have

periplasmic regions. By a bioinformatics evaluation, of those four

GGDEF proteins that increased biofilm formation, only PA1107

and TpbB have transmembrane regions. Taken together with the

results of genetic screening, TpbB is the most likely target protein

for TpbA. Also, our results imply the periplasmic Y48 and Y62

residues of TpbB are the likely targets for tyrosine phosphoryla-

tion. We are now investigating whether TpbA regulates the

activity of GGDEF proteins to control cellular c-di-GMP

concentrations in P. aeruginosa.

The relationship between tyrosine phosphorylation and biofilm

formation is not well established. We found that trisodium

orthovanadate treatment increased biofilm formation of PA14

(Fig. S4), indicating that tyrosine phosphorylation increases biofilm

formation in P. aeruginosa. Recently, Ltp1, a low molecular weight

tyrosine phosphatase in non-motile, Gram-negative P. gingivalis,

was identified as a negative regulator of EPS production and

biofilm formation [45]. A sequence similarity search shows TpbA

is not a homolog of Ltp1, because TpbA has a signal sequence in

its N-terminal region, and TpbA is translocated into the periplasm.

Other differences were found in the position of the motif for the

tyrosine phosphatase, since TpbA has the motif at the position 132

and Lpt1 has it at position 9. It appears the P. aeruginosa genome

encodes another tyrosine phosphatase, annotated as ptpA, that has

a tyrosine phosphatase motif at the position 7 and does not have a

signal sequence at the N-terminus. The function of PtpA is

unknown but it is essential [46].

In contrast to poorly-investigated Tyr phosphorylation, regula-

tion of biofilm formation by phosphorylation has been identified for

several systems; for example, for the His kinase/Asp response

regulator phosphorylation systems RocS1/RocA1/RocR of P.

aeruginosa PAK [47] and the PAO1 PA1611/PA1976/PA2824/

RetS/HptB system of P. aeruginosa [48]. In addition, in the B. subtilis

PrkC/PrpC system [18], loss of a membrane-anchored Thr kinase

and its phosphatase reduces biofilm formation. Our results indicate

that TpbA acts as a negative regulator of cellular c-di-GMP

formation and loss of TpbA results in increased c-di-GMP

concentrations that enhance biofilm formation and inhibit motility.

These results show clearly that posttranslational modification

through phosphatase activity is related to bacterial biofilm

formation as well as to the regulation of the synthesis of cellular

second messengers. In addition, by showing tpbA transcription is

increased by LasR (Fig. 7) and by finding AHL-binding motifs, we

have now linked quorum sensing to c-di-GMP concentrations and

biofilm formation in P. aeruginosa. Similarly, Vibrio cholerae QS was

found recently to reduce cellular c-di-GMP concentrations via a c-

di-GMP-specific phosphodiesterase which leads to lower biofilm

formation [49]. A common element in both studies is that QS seems

to be a negative regulator of c-di-GMP. The tpbA mutation caused a

hyper-aggregative phenotype (Fig. 4), and this would lead to flat

biofilms since the wspF mutant, which accumulates increased c-di-

GMP, formed flat biofilms [25]. Formation of flat and undifferen-

tiated biofilms is also observed by loss of LasI function [12] that can

activate tpbA expression. Hence, TpbA might participate in

developing biofilm structure. These results are important in that

the regulatory networks that control c-di-GMP concentrations are

now linked to the environment and cell populations.

Materials and Methods

Bacterial strains and growth conditionsStrains used in this study are listed in Table 2. P. aeruginosa PA14

wild-type and its isogenic mutants were obtained from the

Harvard Medical School [46]. Transposon insertion of the tpbA

mutant was verified as described previously with a minor

modification [50]. Briefly, the tpbA gene was amplified from

chromosomal DNA using primers PA14_13660-VF and

PA14_13660-VR (Table S3) which did not amplify chromosomal

DNA from the tpbA mutant. In addition, the DNA fragment

corresponding to the end of the transposon and tpbA gene was

amplified with tpbA chromosomal DNA using primers

PA14_13660-VF and GB-3a (Table S3) and PA14_13660-VR

and R1 (Table S3) but these pairs of primers did not amplify PA14

wild-type chromosomal DNA. P. aeruginosa and E. coli were

routinely grown in Luria-Bertani (LB) medium at 37uC unless

noted. Gentamicin (15 mg/mL) and tetracycline (75 mg/mL) were



Table 2. Strains used in this study.

Strain Genotype or description Reference

P. aeruginosa

PA14 Wild-type strain [46]

PA14_13660 (PA3885, tpbA) PA14_13660 V Mar2xT7, GmR [46]

PA14_19120 (PA3477, rhlR) PA14_19120 V Mar2xT7, GmR [46]

PA14_50360 (PA1086, flgK) PA14_50360 V Mar2xT7, GmR [46]

PA14_49880 (PA1121) PA14_49880 V Mar2xT7, GmR [46]

PA14_49890 (PA1120, tpbB) PA14_49890 V Mar2xT7, GmR [46]



PA14_24480 (PA3064, pelA) PA14_24480 V Mar2xT7, GmR [46]

PA14_24490 (PA3063, pelB) PA14_24490 V Mar2xT7, GmR [46]

PA14_24510 (PA3061, pelD) PA14_24510 V Mar2xT7, GmR [46]

PA14_24560 (PA3058, pelG) PA14_24560 V Mar2xT7, GmR [46]



PA14_45940 (PA1432, lasI) PA14_45940 V Mar2xT7, GmR [46]

PA14_19130 (PA3476, rhlI) PA14_19130 V Mar2xT7, GmR [46]

PA14_lasR rhlR (PA1431 PA3477, lasR rhlR) PA14 DlasR DrhlR [70]

E. coli

BL21(DE3) F2 ompT hsdSB(rB2mB

2) gal dcm l(DE3) V placUV5:: T7 polymerase Novagen

HB101 pro leu thi lacY Strr endoI2 recA2 r2 m2 [67]

S17-1(lpir)/pUT-miniTn5-luxAB TcR SmR TpR mod+ res thi pro recA hsdR17 V RP4-TC::Mu-Km::Tn7 with pUT-miniTn5-luxAB [59,71]

TG1 K12, lac–pro supE thi hsdD5 (F9 traD36 proA+B+ lacIq lacZ M15) [61]

AG1 recA1 endA1 gyrA96 thi-1 hsdR17(rK2 mK

2) supE44 relA1 [72]

Plasmids

pMQ70 CarR, ApR, PBAD, expression vector [51]

pMQ70-tpbA CarR, ApR, PBAD::tpbA, complementation plasmid This study

pMQ70-tpbB CarR, ApR, PBAD::tpbB, complementation plasmid This study

pMQ70-tpbB-Y48F CarR, ApR, PBAD::tpbB, Y48 of TpbB replaced with F48 This study



pRK2013 Mobilizing conjugation plasmid [68]

pET28b KmR, PT7 expression vector Novagen

pET28b-13660n KmR, PT7::tpbA-nHis+, expression vector for TpbA-nHis This study

pET28b-13660c KmR, PT7::tpbA-cHis+, expression vector for TpbA-cHis This study

pLP170 CarR ApR, promoterless-lacZ [73]

pLP-ptpbA CarR ApR, ptpbA::lacZ This study

pPCS1001 CarR ApR, plasR::lacZ [73]

pPCS1002 CarR ApR, prhlR::lacZ [73]

pCA24N CmR, lacIq, pCA24N [72]

pCA24N-yddV CmR, lacIq, pCA24N PT5-lac::yddV+ [72]

pCA24N-cpdB CmR, lacIq, pCA24N PT5-lac::cpdB+ [72]

pCA24N-oxyR CmR, lacIq, pCA24N PT5-lac::oxyR+ [72]

pGEM-T easy CarR ApR, TA cloning vector Qiagen

GmR, TcR, KmR, CarR, CmR, and ApR indicate gentamicin, tetracycline, kanamycin, carbenicillin, chloramphenicol, and ampicillin resistance, respectively.doi:10.1371/journal.ppat.1000483.t002



used for growth of the P. aeruginosa transposon mutants,

carbenicillin (300 mg/mL) was used to maintain P. aeruginosa

plasmids, and kanamycin (50 mg/mL) and chloramphenicol

(50 mg/mL) were used to maintain E. coli plasmids (Table 2).

Complementation of P. aeruginosa mutantsFor complementation of the tpbA and tpbB mutations, tpbA and

tpbB were expressed under the control of the pBAD promoter in

pMQ70 [51]. tpbA and tpbB were amplified using a Pfu DNA

polymerase with primers PA14_13660-F1-NheI and PA14_13660-

R-cHis-HindIII and PA14_49890-F1-NheI and PA14_49890-R-

cHis-HindIII, respectively (Table S3). PCR products were cloned

into the NheI and HindIII sites of pMQ70. The resulting

plasmids, pMQ70-tpbA and pMQ70-tpbB, were transformed into

PA14 and the mutants by conjugation. Briefly, 1 mL of overnight

culture of the recipient strain (PA14 or the mutant), helper strain

(HB101/pRK2013), and donor strain (TG1/pMQ70, TG1/

pMQ70-tpbA, or pMQ70-tpbB) was washed with 1 mL of fresh

LB medium. The mixture of three strains was incubated on LB

plates at 37uC overnight. PA14 strains with pMQ70-based plasmid

were selected on LB plates with 100 mg/mL rifampicin (to kill the

donor and helper), 300 mg/mL carbenicillin (to kill P. aeruginosa

without pMQ70-based plasmids), and 15 mg/mL gentamicin (if a

recipient was a PA14 mutant constructed using a transposon

insertion with the GmR gene). If indicated, 0.05% arabinose was

added to induce gene expression.

Biofilm formationBiofilm formation was examined in 96-well polystyrene plates

using crystal violet staining [52]. Overnight cultures of P. aeruginosa

were diluted to a turbidity of 0.05 at 600 nm with fresh LB

medium, and then 150 mL of diluted bacterial culture was

incubated in 96-well polystyrene plates for 2, 4, 8, 24, and 50 h.

Ten wells were used for each strain and three independent cultures

were used for each experiment. Trisodium orthovanadate, a

tyrosine phosphatase-specific inhibitor, was added to LB medium

at 10 mM.

Colony morphologyTo observe colony morphology, overnight cultures were diluted

to a turbidity of 0.005 at 600 nm with T-broth (10 g/L tryptone),

and 2 mL of diluted cultures were spotted on Congo-red plates

(10 g/L tryptone, 40 mg/mL Congo-red, and 20 mg/mL Coo-

massie brilliant blue) [3]. Plates were incubated at 37uC or room

temperature for 3 to 7 days.

Motility assaysSwimming motility was examined with cells grown to a turbidity

of 1 at 600 nm using 0.3% agar plates with 1% tryptone and

0.25% NaCl [53] and swarming motility was examined with BM-2

plates (62 mM potassium phosphate, 2 mM MgSO4, 10 mM

FeSO4, 0.1% casamino acid, 0.4% glucose, and 0.5% Bacto agar)

[54]. Motility was measured after 24 h. Five plates were tested for

each culture, and two independent cultures were used. The flgK

[22] and rhlR [23] mutants were used as negative controls for

swimming and swarming, respectively.

Aggregation assayAggregation was examined by diluting overnight cultures with

fresh LB medium in 5 mL screw-capped tubes from 0% (no added

fresh LB medium) to 100% (pure fresh LB medium). Cells were

inverted gently several times and placed at room temperature for

15 min.

Pellicle formationOvernight cultures of PA14, the tpbA mutant, and the pelA

mutant were diluted to a turbidity of 0.005 at 600 nm in 4 mL T-

broth, and the bacterial cultures were placed in a polycarbonate

glass tube at 37uC or room temperature [3].

EPS assayPel-dependent EPS production was quantified as described

previously [6] based on the amount of Congo red that binds to the

EPS. Briefly, 1 mL of overnight culture was washed with 1 mL T-

broth. Due to aggregative phenotype of the tpbA mutant, cell

pellets of the tpbA mutant, wild-type, and pelA mutant (negative

control) were sonicated three times at 3W for 10 sec. Bacterial

suspensions in T-broth (500 mL) were incubated with 40 mg/mL

Congo-red at 37uC or room temperature with vigorous shaking.

After 2 h, the absorbance of the supernatants of the each

suspension was measured at 490 nm using a spectrophotometer.

T-broth with 40 mg/mL Congo-red was used as a blank.

Rhamnolipids assayProduction of rhamnolipids was determined as described

previously [55]. Overnight cultures were diluted to a turbidity of

0.05 at 600 nm in 25 mL LB medium and were re-grown at

250 rpm for 24 h to eliminate the effect of antibiotics. The

supernatants of the bacterial cultures were used to determine the

relative concentrations of rhamnolipids using orcinol/sulfuric acid.

Rhamnose (Fisher Scientific, Pittsburgh, PA) was used as a standard.

Whole-transcriptome analysisThe P. aeruginosa genome array (Affymetrix, P/N 510596) was

used to investigate differential gene expression in biofilm cells

between PA14 and the tpbA mutant. Biofilm cells were harvested

from 10 g of glass wool [56] after incubation for 4 h and 7 h in LB

with shaking at 250 rpm, and RNA was extracted with a RNeasy

Mini Kit (Qiagen) [57]; note the RNase inhibitor RNAlater

(Applied Biosystems, Austin, TX) was used for the 4 h and second

7 h set of microarrays. Global scaling was applied so the average

signal intensity was 500. The probe array images were inspected

for any image artifact. Background values, noise values, and

scaling factors of both arrays were examined and were

comparable. The intensities of polyadenosine RNA controls were

used to monitor the labeling process. If the gene with the larger

transcription rate did not have a consistent transcription rate based

on the 13 probe pairs (p-value less than 0.05), these genes were

discarded. A gene was considered differentially expressed when the

p-value for comparing two chips was lower than 0.05 (to assure

that the change in gene expression was statistically significant and

that false positives arise less than 5%) and when the expression

ratio was higher than the standard deviation for the whole

microarrays [58], 1.4 for 4 h, 1.7 for the first 7 h replicate, and 2.2

for second 7 h replicate. All three sets of whole-transcriptome data

were deposited at the Gene Expression Omnibus (GSE13871).

qRT-PCRqRT-PCR was performed using the StepOnePlusTM Real-Time

PCR System (Applied Biosystems, Foster City, CA). Expression of

pelA, the PA4625 gene, and the PA4139 gene was determined

using total RNA isolated from two independent biofilm cultures of

PA14 and the tpbA mutant. The biofilm cells were grown and total

RNA were isolated in the same manner as described above for the

whole-transcriptome analysis. The primers for qRT-PCR are

listed in Table S3. The housekeeping gene rplU [44] was used to

normalize the gene expression data.



Genetic screeningTo isolate the suppressive loci for TpbA functions, a double

mutant library was generated using the Tn5-luxAB transposon with

the background of the tpbA mutation as described previously [59].

Briefly, 1 mL of overnight culture of the P. aeruginosa tpbA mutant

and E. coli S17-1 (lpir) with Tn5-luxAB were grown on LB plates

together overnight. Cells were harvested from the plate and

resuspended in 10 mL of LB medium. Screening of cells with

mutations in addition to tpbA was performed in three steps.

Suppression of the highly-aggregative phenotype of the tpbA

mutant was used first; the cell mixture (P. aeruginosa single and

double mutants along with E. coli S17-1) was placed at room

temperature for 15 min and the supernatant was used for

secondary screening (cells with the tpbA mutant aggregative

phenotype were therefore discarded). Supernatant cells were

spread on Congo-red plates with 50 mg/mL gentamicin (to kill E.

coli), and 75 mg/mL tetracycline (to kill the tpbA single mutant),

and incubated for 3–4 days. P. aeruginosa double mutants with

smooth surfaces were picked (the tpbA mutant was red and

wrinkled). The crystal violet biofilm assay was used for the third

screening, and mutants showing decreased biofilm formation in

comparison to that of the tpbA mutant were chosen as phenotype

reversal mutants. The insertion position of Tn5-luxAB transposon

was determined by two-step PCR as described previously [59] with

primers LuxAB inside and Arb1 for the first round of PCR and

LuxAB outside and Arb2 for the second round of PCR (Table S3).

The PCR product was ligated into pGEM-T easy (Promega,

Madison, MI) and sequenced using a BigDye Terminator Cycle

Sequencing Kit (Applied Biosystems, Foster City, CA).

Quantification of c-di-GMPc-di-GMP was isolated as described previously [60]. P. aeruginosa

was grown in 1 L of LB medium for 16 h at 250 rpm, and

formaldehyde (final concentration of 0.18%) was added to

inactivate degradation of c-di-GMP. Cells were harvested by

centrifugation at 8,000 g for 10 min at 4uC. Nucleotide extract

was prepared as described previously [60]. Cell pellets were

washed with 40 mL of phosphate buffered saline (pH 7) [61] with

0.18% formaldehyde and centrifuged at 8,000 g for 10 min at 4uC.

The cell pellets were dissolved in H2O and boiled for 10 min.

After cooling the samples on ice for 10 min, nucleotides were

extracted in 65% ethanol. Supernatants were transferred, and the

extraction was repeated. Pooled supernatants were lyophilized,

and pellets were dissolved in 1 mL of 0.15 M triethyl ammonium

acetate (TEAA, pH 5.0). The samples were filtered using a PVDF

filter (0.22 mm), and 20 mL of each sample was fractionated using

HPLC (Waters 515 with photodiode array detector, Milford, MA)

with a reverse-phase column (Nova-PakH C18 column; Waters,

15063.9 cm, 4 mm). Separations were conducted in 0.15 M

TEAA at a 1 mL/min flow rate using gradient elution with

acetonitrile (0% to 15% concentration). Synthetic c-di-GMP

(BIOLOG Life Science Institute, Bremen, Germany) was used as

a standard. The peak corresponding to c-di-GMP from the extract

of the tpbA mutant was verified by co-elution with standard c-di-

GMP. E. coli AG1/pCA24N-yddV that has an elevated c-di-GMP

concentration [62] was also used as a positive control.

Plasmid construction of pET28b-13660c and purificationof recombinant TpbA-cHis

To determine if TpbA is a phosphatase, tpbA was amplified with

a Pfu DNA polymerase using primers PA14_13660-F-XbaI and

PA14_13660-R-XhoI (Table S3). The PCR product was digested

with XbaI and XhoI and was ligated in-frame to the polyhistidine

tag sequence of the pET28b vector. The resulting plasmid,

pET28b-13660c has the tpbA gene fused to a 66His tag at the C-

terminus (TpbA-cHis) and under control of the T7 promoter. The

pET28b-13660c plasmid was confirmed by DNA sequencing with

the T7 promoter and T7 terminator primers (Table S3).

Production of TpbA-cHis was induced in E. coli BL21(DE3) cells

with 1 mM IPTG at room temperature overnight. TpbA-cHis was

purified using a Ni-NTA resin (Qiagen, Valencia, CA) as

described in a manufacturer’s protocol. Purified TpbA-cHis was

dialyzed against buffer (50 mM Tris-HCl, 100 mM NaCl, 10%

glycerol, 0.01% Triton X-100, pH 7.5) at 4uC overnight.

Phosphatase assayThe p-nitrophenyl phosphate assay (pNPP) was used to examine

TpbA-cHis phosphatase activity [63]. Purified TpbA-cHis protein

was incubated in 100 mL of reaction buffer (50 mM Tris-acetate,

10 mM MgCl2, 10 mM pNPP, 5 mM DTT, pH 5.5) at 37uC for

1 h. The reaction was quenched by adding 900 mL of 1 M NaOH.

Trisodium orthovanadate, a specific inhibitor for tyrosine

phosphatase [30], was used at 10 mM. p-nitrophenol was

measured at an absorbance of 405 nm. An extinction coefficient

of 1.786104 M21 cm21 was used to calculate the concentration of

p-nitrophenol.

To examine if TpbA is a tyrosine specific phosphatase, a

tyrosine phosphatase assay was performed using the Tyrosine

Phosphatase Assay System (Qiagen). Eight micrograms of TpbA-

cHis were incubated with either 50 mM phosphotyrosine peptide

type I (END(pY)INASL) or peptide type II (DADE(pY)LIPQQG)

in a reaction buffer (50 mM Tris-Acetate, 10 mM MgCl2, pH 5.5)

at 37uC for 3 h. The reaction was quenched using a molybdate

dye solution and incubated for 30 min at room temperature.

Released phosphate was quantified by measuring the absorbance

at 630 nm.

Determination of the subcellular localization of TpbATpbA-cHis protein was expressed in BL21(DE3) cells with

1 mM IPTG for 4 h at 37uC. Periplasmic proteins were purified

using a PeriPreps Periplasting Kit (Epicentre Technologies,

Madison, WI) as well as cytoplasmic and membrane proteins.

Escherichia coli CpdB [64] was used as a control of periplasmic

protein and the E. coli OxyR [65] was used for the cytoplasmic

control. Fractionated proteins as well as TpbB were analyzed by

12% SDS-PAGE.

Site-directed mutagenesis of the TpbB periplasmictyrosine residues

Site-directed mutagenesis of the predicted periplasmic tyrosine

residues of TpbB was performed to convert them to phenylalanine

(Y48F, Y62F, and Y95F); it was reasoned that phenylalanine

would provide a similar bulky side chain but remove the hydroxyl

moiety needed for phosphorylation [66]. The mutations were

introduced into pMQ70-tpbB using Pfu DNA polymerase and

QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla,

CA), and the primers are listed in Table S3. The resulting

plasmids, pMQ70-tpbB-Y48F, pMQ70-tpbB-Y62F, and pMQ70-

tpbB-Y95F were transformed into the tpbB mutant by conjugation

and aggregation was assayed. DNA sequencing was used to

confirm the tyrosine mutations and that no other mutations were

introduced into the promoter or protein-coding sequences.

b-galactosidase activityThe promoter region of tpbA (ptpbA), including 399 bp upstream

of the start codon and 31 bp of the open reading frame, was



amplified using Pfu DNA polymerase with primers pPA14_13660-

F-HindIII and pPA14_13660-R-BamHI (Table S3). The PCR

product (430 bp) was cloned into the HindIII/BamHI sites of

pLP170 to produce pLP-ptpbA, and it was conjugated into PA14

and the QS-related mutants using helper strain HB101/pRK2013

[67,68]. Transformants were grown overnight in LB medium with

300 mg/mL carbenicillin, reinoculated at a turbidity of 0.05 at

600 nm, and grown for another 6 h. Biofilm cells were harvested

from 4 g of glass wool after incubation for 6 h in LB at 37uC with

shaking at 250 rpm. b-galactosidase activity was measured using

suspension cells and biofilm cells as described previously [69].

Similarly, b-galactosidase activity of plasR::lacZ (pPCS1001) and

prhlR::lacZ (pPCS1002) was examined in PA14 and the tpbA

mutant.

Supporting Information

Figure S1 Complementation of the tpbA mutant using biofilm

formation and aggregation. Total biofilm formation (A) and

bottom biofilm formation on polystyrene plates (B) by P. aeruginosa

PA14 and the tpbA mutant with either pMQ70 or pMQ70-tpbA in

LB with 300 mg/mL carbenicillin and 0.05% arabinose after 24 h

at 37uC. Six wells were used for each culture. Data show the

average of the two to four independent experiments6s.d. Cell

aggregation of PA14 and the tpbA mutant with either pMQ70 or

pMQ70-tpbA (C). Overnight cultures (1 mL) were mixed with

3 mL of fresh LB medium, and the tubes were placed at room

temperature for 15 min.

Found at: doi:10.1371/journal.ppat.1000483.s001 (2.54 MB TIF)

Figure S2 Complementation of the tpbB mutant using aggrega-

tion. Bacterial cultures were grown at 37uC at 250 rpm overnight.


Figure S3 Quantification of cellular c-di-GMP concentrations

by HPLC from 30 mg of cells. 10 mM synthetic c-di-GMP (A),

nucleotide extract from PA14 (B), nucleotide extract from the tpbA

mutant (C), and nucleotide extract from the tpbA mutant spiked

with 10 mM c-di-GMP (D). Spectra of synthetic c-di-GMP (E) and

nucleotide extract from the tpbA mutant (F).


Figure S4 Normalized biofilm formation of PA14 at 37uC in LB

after 24 h with and without tyrosine phosphatase inhibitor

Na3VO4 (10 mM). Six wells were used for each culture. Data

show the average of the two independent experiments6s.d.


Figure S5 Aggregation is reduced by site-directed mutagenesis

at Y48 and Y62 of TpbB. The tpbB mutant was grown in 25 mL of

LB medium supplemented with carbenicillin (300 mg/mL) and

gentamicin (15 mg/mL) with pMQ70 (negative control) and

pMQ70-tpbB (positive control) (A), pMQ70-tpbB-Y48F (B),

pMQ70-tpbB-Y62F (C) and pMQ70-tpbB-Y95F (data not shown).

Percentage of cultures with each phenotype (aggregation or no

aggregation are indicated). A total of 46 and 37 independent

cultures was tested for tpbB/pMQ70-tpbB-Y48F and tpbB/

pMQ70-tpbB-Y62F, respectively, and representative images are

shown. Note that aggregates were always formed with tpbB/

pMQ70-tpbB (positive control) and tpbB/pMQ70-tpbB-Y95F, but

not with tpbB/pMQ70 (negative control).


Table S1 Partial list of induced genes in biofilm cells in LB

medium after 4 and 7 h at 37uC for the tpbA mutant versus wild-

type PA14 using three sets of DNA microarrays (note RNAlater

was used for the 4 h set and the second set at 7 h).

Found at: doi:10.1371/journal.ppat.1000483.s006 (0.07 MB

DOC)

Table S2 Partial list of repressed genes in biofilm cells in LB

medium after 4 and 7 h at 37uC for the tpbA mutant versus wild-

type PA14 using three sets of DNA microarrays (note RNAlater

was used for the 4 h set and the second set at 7 h).


DOC)

Table S3 Primers used in this study.


DOC)

Acknowledgments

We are grateful to Professor Frederick Ausubel for the PA14 transposon

mutant library, Professor Barbara Iglewski for the pLP170 plasmid,

Professor Marvin Whiteley for Tn5-luxAB, Professor You-Hee Cho for the

PA14 lasR rhlR mutant, and the National Institute of Genetics for the

ASKA clones.

Author Contributions

Conceived and designed the experiments: AU TKW. Performed the

experiments: AU. Analyzed the data: AU TKW. Wrote the paper: AU

TKW.

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connecting quorum sensing, c-di-gmp, pel …...quorum sensing signals, that it is in the periplasm,...

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